Oligodendrocytes, which make the myelin sheaths in central nervous system (CNS), evolve from multipotential neural stem cells through a series of developmental stages (Rogister et al. 1999; Shihabuddin et al. 1999; Levine et al. 2001) for recent reviews). Recognized stages include early bipolar progenitors A2B5+ cells, or O-2A (Raff 1989), late multipolar progenitors expressing the O4 sulfatide glycosides (Schachner et al. 1981), arborized immature oligodendrocytes O4 and GalC positive, and mature oligodendrocytes having the O1 sulfatide and synthesizing the myelin membrane with its structural components such as myelin basic protein (MBP).
Embryonic stem (ES) cell lines, derived from the inner cell mass of blastocyst-stage embryos, are a potential large scale source of oligodendrocytes and precursors derived from murine ES cell have been used for transplantion into myelin deficient CNS (Brustle et al. 1997; Brustle et al. 1999; McDonald et al. 1999). A number of culture conditions have been defined under which murine ES cells differentiated into floating embryoid bodies (EB) may be directed toward the neural lineages giving rise to various types of neurons, to astrocytes and to oligodendrocytes. One approach is based on selection in serum-free defined medium in which neural precursor cells survive, proliferate under the influence of basic fibroblast growth factor (FGF-2) and differentiate upon growth factor removal and plating on adherent substrates (Okabe et al. 1996). Under these conditions, some O4 positive cells develop provided tri-iodothyronine (T3) is added, in line with T3 effect on optic nerve derived O-2A progenitors (Barres et al. 1994). A more efficient selection is obtained by sequential treatment of EB cells by FGF-2, then FGF-2 with epidermal growth factor (EGF), and FGF-2 with Platelet derived growth factor PDGF-AA, a factor promoting proliferation of glial precursor cells (Besnard et al. 1987; Bogler et al. 1990), thereby increasing the number of A2B5+ cells which after growth factors withdrawal differentiate into both O4+ oligodendrocytes and astrocytes expressing glial fibrillary acidic protein GFAP (Brustle et al. 1999).
Another approach uses differentiation agents such as retinoic acid to induce neural and glial lineages in EB cultures (Bain et al. 1995; Fraichard et al. 1995). As in newborn brain derived cultures, neural precursors can be further enriched by selecting non-adherent cells growing as floating spheres in defined medium, and expanding them as neurospheres and oligodendrocyte-enriched oligospheres that differentiate after EGF, FGF removal (Zhang et al. 1998; Liu et al. 2000). Human ES cell lines derived EBs also form neural tube like rosettes expandable as floating neurospheres that can be transplanted in vivo or plated on polycationic substrates to differentiate into neurons, astrocytes and oligodendrocytes, the latter developing particularly after treatment with PDGF-AA and T3 (Reubinoff et al. 2001; Zhang et al. 2001). Although cytokines such as leukemia inhibitory factor (LIF) are known to maintain ES cells in an undifferentiated, multipotent state, it was recently found that LIF allows sparse murine ES cell cultures to develop into neurospheres (Tropepe et al. 2001).
Enriched murine ES cell derived oligospheres, yielding over 90% oligodendrocytes, have been obtained in a complex medium including combinations of hormones, such as T3 and progesterone, and cytokines such as Neurotrophin-3 (NT3) and ciliary neurotrophic factor CNTF (Liu et al. 2000). Both cytokines may contribute to an effect on oligodendrocyte precursors, as observed in optic nerve (Barres et al. 1994; Barres et al. 1996). However, the effects of CNTF on oligodendrocyte differentiation are not clear as in some conditions it mainly induces GFAP+ astrocytes from A2B5+ progenitors (or earlier glioblasts) with little effect on O4+ cells (Lillien et al. 1990; Gard et al. 1995; Johe et al. 1996; Bonni et al. 1997), whereas in other conditions it also increases survival and proportion of GalC+, O1+ and MBP+ cells in the cultures (Kahn et al. 1994; Mayer et al. 1994; Marmur et al. 1998).
CNTF belongs to the interleukin-6 (IL-6) family of cytokines that signal via gp130 either as a heterodimeric receptor with LIF-R (for CNTF, LIF, and oncostatin-M (OSM)) or as a homodimer (for interleukin-6 (IL-6), interleukin-11 (IL-11)) (Taga et al. 1997) for review). There is growing evidence on the importance of gp130 signaling for myelinating cells. In mice, postnatal gene deletion has indicated that gp130 is required to maintain Schwann cell function and myelination in peripheral nerves, in addition to its role in astrocytosis (Betz et al. 1998; Nakashima et al. 1999). With the help of a potent gp130 activating ligand, the IL6R/IL6 chimera in which IL-6 is fused to the extracellular portion of the IL-6 receptor (Chebath et al. 1997), we have previously observed induction of myelin gene expression in embryonic Schwann cells (Haggiag et al. 1999; Haggiag et al. 2001) and activation of myelin gene promoters (Slutsky et al. 2003). Activation of a transgenic MBP gene promoter in mice brain cultures was observed in response to CNTF (Stankoff et al. 2002) and in similar cortical cultures from newborn rat IL6R/IL6 chimera was more effective than CNTF to increase the development of highly arborized GalC+ oligodendrocytes (Valerio et al. 2002).
Promising results have been recently obtained in mice where injections of neural stem cells from the periventricular zone of adult mice brain and grown into neurospheres have induced clinical recovery and remyelination in an animal model of multiple sclerosis (Pluchino et al. 2003). Applying such technology to human patients suffering from multiple sclerosis or other demyelinating diseases, poses many difficulties because the neural stem cells would have to be isolated from cadavers or from aborted fetuses. Hence, the amount of cells that could be obtained would be limited, it would be difficult to ascertain that the brain cells do not transfer dangerous pathogens, and the transplants may cause problems of immuno-histocompatibility and may be rejected.
As indicated, blastocyst-derived ES cell lines, that are indefinitely expandable in laboratory tissue culture conditions, could provide a large-scale source of developing oligodendrocytes capable of myelinating neurons and thereby repairing lesions in the CNS (Cao et al. 2002; Gottlieb 2002).
Therefore, there is a need for a method to promote oligodendrocyte generation from ES cell lines.